Isolation and molecular analysis of the gene for cytochrome c1 from Kluyveromyces lactis

By ethyl methanesulphonate mutagenesis of the yeast Kluyveromyces lactis we have isolated five nuclear mutants that were unable to grow on non-fermentable carbon sources. The mutations were found to belong to three complementation groups. After functional complementation of the mutation in one of these mutants we have cloned the structural gene for cytochrome c ₁, named KlCYT1. This gene has been assigned to chromosome VI and its nucleotide sequence exhibited 74.3% identity to the homologous gene of S. cerevisiae. Received: 6 February 1996 / 2 April 1996     

Humoral immune response to membrane components of Chlamydia trachomatis and expression of human 60 kDa heat shock protein in follicular fluid of in-vitro fertilization patients

Recent evidence suggests that Chlamydia trachomatis can persist in the female upper genital tract in an unculturable state. Since unsuspected C. trachomatis infection has been associated with adverse in-vitro fertilization (IVF) outcome we sought to detect further evidence of C. trachomatis in the genital tracts of women undergoing IVF. The prevalence and distribution of antibodies to the major structural proteins of C. trachomatis in paired follicular fluid and sera of women undergoing IVF were examined. Sera and follicular fluid samples from 149 women were assayed for immunoglobulin (Ig)G and IgA antibodies to two C. trachomatis antigens, the major outer membrane protein (MOMP) and a recombinant lipopolysaccharide (rLPS) fragment. Additionally, the expression of human 60 kDa heat shock protein (hsp 60) in follicular fluid was determined. All cervical and follicular fluid samples were negative for C. trachomatis by polymerase chain reaction, ligase chain reaction and DNA probe. Sera from 60% of the subjects were positive for antichlamydial rLPS IgG; 36% were positive for anti-MOMP IgG. Similarly, rLPS-directed and MOMP-directed IgA were detected in sera of 34 and 14% of the subjects respectively. IgG antibodies to MOMP and rLPS were detected in 42 and 41% of the follicular fluid examined respectively. Anti-MOMP IgA was identified in 8.7% of the follicular fluid while 27.5% were positive for anti-rLPS IgA. Human hsp 60 expression was documented in 11.6% of the follicular fluid tested. IgA antibodies to both MOMP (P = 0.03) and rLPS (P = 0.02) in follicular fluid were associated with a failure to become pregnant after embryo transfer. IgG antibodies in sera and follicular fluid and IgA antibodies in sera were unrelated to IVF outcome. Similarly only anti- MOMP IgA (P = 0.02) and anti-rLPS IgA (P = 0.04) in follicular fluid were correlated with human hsp 60 expression in follicular fluid. The unique association between IgA antibodies to two chlamydial antigens in follicular fluid and both hsp 60 expression and IVF failure provides further support for the possibility that a persistent upper genital tract chlamydial infection contributes to IVF failure in some women.      

Comprehensive Mutation Analysis of PMS2 in a Large Cohort of Probands Suspected of Lynch Syndrome or Constitutional Mismatch Repair Deficiency Syndrome

Monoallelic PMS2 germline mutations cause 5%–15% of Lynch syndrome, a midlife cancer predisposition, whereas biallelic PMS2 mutations cause approximately 60% of constitutional mismatch repair deficiency (CMMRD), a rare childhood cancer syndrome. Recently improved DNA‐ and RNA‐based strategies are applied to overcome problematic PMS2 mutation analysis due to the presence of pseudogenes and frequent gene conversion events. Here, we determined PMS2 mutation detection yield and mutation spectrum in a nationwide cohort of 396 probands. Furthermore, we studied concordance between tumor IHC/MSI (immunohistochemistry/microsatellite instability) profile and mutation carrier state. Overall, we found 52 different pathogenic PMS2 variants explaining 121 Lynch syndrome and nine CMMRD patients. In vitro mismatch repair assays suggested pathogenicity for three missense variants. Ninety‐one PMS2 mutation carriers (70%) showed isolated loss of PMS2 in their tumors, for 31 (24%) no or inconclusive IHC was available, and eight carriers (6%) showed discordant IHC (presence of PMS2 or loss of both MLH1 and PMS2). Ten cases with isolated PMS2 loss (10%; 10/97) harbored MLH1 mutations. We confirmed that recently improved mutation analysis provides a high yield of PMS2 mutations in patients with isolated loss of PMS2 expression. Application of universal tumor prescreening methods will however miss some PMS2 germline mutation carriers.     

Assessing effects of PEEP and global expiratory lung volume on regional electrical impedance tomography

Objective: This study was performed to assess the value of electrical impedance tomography (EIT) as an indicator of tidal (V_T) and end expiratory lung volume (EELV).

Methods: EIT measurements were performed in seven healthy piglets during constant tidal volume ventilation at incremental and decremental positive end-expiratory pressure (PEEP) levels. Tidal impedance changes were calibrated to volume using V_T calculated from flow at the airway opening. Simultaneously, calibrated respiratory inductive plethysmography was used to measure EELV changes, and used as a reference standard.

Results: EIT systematically underestimated both V_T and EELV changes when EELV deviated from the level at which it was calibrated. Calculated over the entire pressure–volume curve, EIT systematically underestimated V_T by 28 ml, with a precision from ?16 to 72 ml. EELV was systemically underestimated by 406 ml, with a precision of ?38 to 849 ml. Nonlinear recruitment in the ventral regions of the lungs was the main cause of this underestimation.

Conclusions: Tidal and end-expiratory changes in pulmonary impedance reflect corresponding changes in lung volume, but the increasing underestimation with increasing lung volume should be taken into account in the analysis of EIT data.     

Case assignment in agrammatism.

Agrammatic speech is characterized by the omission and substitution of grammatical morphemes. Some recent papers suggest that certain patterns of omission and substitution are ruled by linguistic, that is, syntactic processes (e.g., Hagiwara, 1995; Friedmann & Grodzinsky, 1997; Bastiaanse & Van Zonneveld, 1998). In the present paper, the omission pattern of case markers in the spontaneous speech of Dutch and German speakers with agrammatic aphasia is analyzed within the framework of Chomsky's (1986) case theory, which says that every phonetically realized NP must receive (abstract) case. The inflected verb (I) assigns nominative case to the subject in the sentence, and the verb (V) assigns dative and accusative case to the indirect and direct object, respectively. This, in combination with the knowledge that verbs and verb inflections are notoriously difficult for speakers with agrammatism, served as the basis for this study. We hypothesize that, if no case assigner is produced, the noun will receive nominative case by default or the case marking morpheme (i.e., the determiner) will be omitted. This hypothesis has been tested and was supported by the data.     

Outcomes and costs of treatment with risperidone in adult and elderly patients: the Delta patient using risperidone study

Purpose: The primary aim of the present study (called DePUR: Delta patients using risperidone) was to compare patient characteristics, treatment outcome variables and medication costs of adult and elderly patients treated with risperidone. Methods: In a single-centre retrospective study, the following data were extracted from the medical records of 32 adult (<65 years) and 32 elderly (≥65 years) inpatients to whom risperidone was prescribed shortly after admission: demographic data, diagnosis, medication and hospitalisation history, all drugs taken during admission, treatment effectiveness of risperidone, reported adverse events. These data were collected for the period from admission until discharge or until they had been hospitalised for 120 days. Results: Most patients (81% of adult patients and 91% of elderly patients) received risperidone for a psychiatric disease other than schizophrenia. In elderly patients, major depressive disorder with psychotic features was the most frequent diagnosis. Treatment with risperidone was in most cases effective (72% and 74%, respectively), but effectiveness was established sooner in the adult patients than in the elderly patients (7 versus 14 days). The length of stay in hospital was shorter in adults than in elderly (38.5 versus 92.7 days). The daily dose of risperidone was higher in adults than in elderly patients (3.6 versus 1.7 mg per day). Contrary to expectations, daily drug cost of risperidone and daily drug cost of all inpatient drugs were not cheaper in adult patients than in the elderly, mainly due to a higher dose of risperidone and a higher consumption of antipsychotic co-medication in adults. Conclusion: Risperidone is prescribed successfully for many other psychotic features than for schizophrenia. Treatment of elderly patients in our clinic does not require higher daily medication costs than for adult patients.     

Autologous transplantation of bone marrow purged in vitro with anti-CD7- (WT1-) ricin A immunotoxin in T-cell lymphoblastic leukemia and lymphoma

Seven patients with high-risk acute T-cell lymphoblastic leukemia (T- ALL) and six with T cell lymphoma (T-LL) were treated with autologous bone marrow transplantation (ABMT) after in vitro purging of their bone marrow with WT1 (CD7)-ricin A-chain immunotoxin. CD7 expression on the tumor cells showed large variations between the individual patients and was highly related to the specific cytotoxicity of WT1-ricin A. Incubation of bone marrow with up to 10(-8)mol/L WT1-ricin A in the presence of 6 mmol/L NH4Cl did not compromise the growth potential of the hematopoietic progenitors CFU-GM, CFU-GEMM, and BFU-E. Hematologic engraftment (greater than 10(9) leukocytes/L) occurred within a normal time period (median, 17 days). Seven patients are alive and in complete remission (CR) at 48+, 44+, 40+, 26+, 11+, 7+, and 6+ months after ABMT. Four patients relapsed within 6 months after ABMT. Two of them had the lowest CD7 expression on their tumor cells, the other two were transplanted in CR2 and CR3. Two patients died from transplantation related infections. The immunologic reconstitution was delayed, although the numbers of T cells reached normal levels within 1 month. The number of CD7+ cells remained low up to 1 year after transplantation. The T4/T8-ratio was decreased for at least 6 months. The T-cell response to mitogens recovered to normal levels after 1 year. This study shows that ABMT with WT1-ricin A purged bone marrow in high-risk T-cell malignancies results in a complete hematopoietic and a delayed immunologic reconstitution. The actuarial relapse free survival is 61% at 3 years.